The present invention relates to trihydroxystilbenes and the glycosylated derivatives thereof. The present invention also relates to methods for isolation and purification of these products using reverse phase liquid chromatography and a method for converting glycosylated to aglycone product. The present invention further relates to treatment of diseases using compounds of the invention.
Resveratrol, 3,4xe2x80x2,5-trihydroxystilbene, was first isolated from grape leaves (Inghim, T. L., Phytochem., 15 (1979) (1976)). Inghim characterized the structure of resveratrol using chemical methods. Resveratrol has following chemical structure in which both R1 and R2 are hydrogen. 
Where xcex2-glc is O-xcex2-D-glucose
Trihydroxystilbenes and derivatives thereof derivatives are reported to have medicinal properties including anti-leukemic and anti-tumor activities. For example, plant material containing resveratrol has been used as an herbal medication for treatment of hyperlupemia and liver diseases in China and Japan for many centuries (Kimura, K. M. et al., Shoygakugaku Zasshi, 83, 35-58 (1981)). Subsequent experiments with purified trans-resveratrol demonstrate that the many biologically useful functions, including modulation of hepatic cholesterol synthesis, inhibition of lypooxygenase activity (Kimura, Y. et al., Biochem. Biophys. Acta. 834, 275 (1985)), inhibition of anaphylactoid (Ragazy, E., et al., Pharmacol. Ref. Commun., 79, 20 (1988)), and protection of lypoproteins against oxydative and free radical damage (Frankel, E. N., et al. Lancet, 1, 1017 (1979)).
Recent literature reports indicate that the extract derived from Cassia quinquangulata Rich (neguminasae) collected in Peru is a potent inhibitor of cyclooxygenase (COX) (Kudo, T. et al., Gann, 71, 260 (1980), Pollard, M. et al., Cancer Lett., 21, (1983), Waddell, W. R. et al., Am. J. Surg., 157, 175 (1989), Thun, M. J. et al., N. Engl. J.Med., 325, 1593 (1991)).
In the following discussion, ED50 represents effective dosage for 50% inhibition.
In a recent article, Meishiang Jang reported that resveratrol inhibits the hydroperoxidase activity of COX-1, ED50=3.7 xcexcM, and also hydroperoxidase activity of COX-2, ED50-85 xcexcM (Jang, M. et al., Science, 275 218 (1997). This inhibitory activity is relevant to cancer therapy and prevention because COX catalyzes the conversion of arachidonic acid to pro-inflammatory substances such as prostaglandins, which can stimulate tumor cell growth and suppress immune responses (Plescia, O. J. et al., Proc. Nat. Acad. Sci. USA., 72, (1975)).
3,4xe2x80x25-Trihydroxystilbene (Resveratrol) has also been found to inhibit certain events associated with tumor growth. For instance, resveratrol inhibits the free radical formation, ED50=27 xcexcM, when human promyclocytic leukemia cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (Shama, S. et al. Cancer Res. 54 5848 (1994). Moreover, Jang et al. investigated the anti-inflammatory activity of resveratrol. In the carrageenan-induced model of inflammation in rats, resveratrol significantly reduced pedal edema both in the acute phase (3 to 24 hours) and the chronic phase (24 to 144 hours). The edema-suppressing activity of resveratrol was greater than that of phenylbutazone and similar to that of indomethacin. Jang et al. also investigated the effect of resveratrol in a mouse mammary gland culture model of carcinogenesis. Resveratrol, in a dose-dependent manner, inhibited the development of DMBA-induced preneoplastic lesions (ED50=3.1 xcexcM). (Jang, M. et al., Science, 275, 218 (1997)).
There has recently been an increase in interest in resveratrol and analogous compounds as a result of epidemiological data showing a lower incidence of mortality due to cardiovascular damage in populations with a high-calorie high-lipid diet, but whose diet also includes red wine, as compared to populations who had a lower calorie consumption and lower percentage of lipids, but whose diet does not include red wine (Seigneur, M. et al. J. Appl. Card., 5, 215 (1990); Siemann, E. H. and Creasy, L. L., Am. J Enol. Vitic. 43, 49 (1992); Renaud, S. and De Lorgeril, M., Lancet, 339, 1523 (1992); Scharp, D., Lancet. 341 27 (1993)).
Investigations have revealed that resveratrol effectively possesses many pharmacological activities which can potentially explain the protective effects of red wine at the cardiovascular level (Frankel, E. N. et al., Lancet, 341 454 (1993)). In addition, resveratrol has proved capable of promoting the formation of nitroxides which have a vasodilatory action and inhibit platelet aggregation induced by collagen or ADP (Fitzpatrick, D. et al., Am. J Physiol., 265 (Heart Circ. Physiol.), 34 774 (1993)).
Plant materials that are natural sources for resveratrols include Vitis vinifera and Polygonum cuspidatum (Huzhang). The concentration of resveratrol in P. cuspidatum is much higher than in V. vinifera. The procedures currently practiced for isolating resveratrols from plant materials are very difficult and low yielding normal phase chromatographic procedures that also use chlorinated solvents which are toxic to humans and can damage the environment.
The isolation of resveratrols from natural sources represents a potential reliable source of supply. The present invention provides an isolation and purification technique which provides high yields and low cost of production of resveratrol and related compounds.
By the present invention, products containing a stilbene fraction, compositions containing these products, and reverse phase liquid chromatography processes for isolating and purifying these products from plant material are identified.
The present invention provides a first product having a solids content of at least about 60% wherein the solids include at least about 10% by weight of a stilbene fraction and a process for making the product that includes the step of contacting a plant material with an alcohol and obtaining the product from the alcohol after contacting.
Also provided is a second product obtained by mixing the first product with a pharmaceutically acceptable processing excipient and drying the resulting mixture.
The present invention further provides a third product made up of at least about 20% by weight of a mixture of trihydroxystilbenes and mono-xcex2-D-glycosylated trihydroxystilbenes and a composition of the third product with an aqueous solvent. According to the present invention, the composition including the third product is made by an MD-1 reverse phase liquid chromatography process.
Similarly, the present invention provides fourth products made up of at least about 30% by weight of a stilbene fraction including trihydroxystilbenes and mono-xcex2-D-glycosylated trihydroxystilbenes and compositions of these fourth products with aqueous solvents. Fourth products are made using an MD-2 process starting with a composition containing the third product in which a polyamide resin is the stationary phase. A composition containing the third product is concentrated to form a loading concentrate that is loaded onto an MD-2 column, optionally using a washing elution volume, followed by elution with one or more MD-2 elution volumes of an aqueous solvent, especially a mixture of an alcohol and water to make an MD-2 effluent that is a composition containing a fourth product. The effluent is collected in toto or as gradient fractions collected by fractionate collection. Fourth products are obtained by removing aqueous solvent from an MD-2 effluent, however collected.
The present invention also provides fifth products that are made up of at least about 60% of a stilbene fraction. A fifth product can be at least about 85% by weight mono-xcex2-D-glycosylated trihydroxystilbenes or at least about 85% aglycone thereof. Also provided are compositions including fifth products and an aqueous solvent. Fifth products are made by an MD-3 process in which the stationary phase is silica gel based. The starting material for an MD-3 process is an effluent, especially a gradient fraction, from an MD-2 process. The effluent is concentrated to form a loading concentrate that is eluted through the MD-3 column and can be followed by a washing elution volume. The MD-3 column is then eluted with one or more elution volumes of an aqueous solvent. Each elution volume can consist of one or more discrete gradient volumes, each made up of a different aqueous solvent, or the composition of each elution volume can vary linearly, exponentially, logarithymically, hyperbolically, or stepwise during elution of the elution volume. The effluent from a first MD-3 process is a composition containing a fifth product. The effluent may be collected in toto or fractionate collected as gradient fractions. Fifth products are obtained by removing aqueous solvent from effluent or gradient fractions of a first MD-3 process. A gradient fraction of a first MD-3 process is the starting material for a cold crystallization process to make a fifth product that contains at least about 85% by weight mono-xcex2-D-glycosylated trihydroxystilbene. The gradient fraction of a first MD-3 process is concentrated to a solids content of at least about 20 g/L and then diluted with water. The resulting mixture is cooled to less than about 0xc2x0 C. to form a slurry from which such fifth product can be isolated, washed, and then dried.
Similarly, the present invention provides a second MD-3 process for making compositions containing sixth products that are at least about 70% by weight, trihydroxystilbenes. Sixth products are isolated by removing aqueous solvent from the compositions. Starting material for a second MD-3 process is an MD-2 gradient fraction that has been fractionate collected. The MD-2 gradient fraction is concentrated to a loading concentrate having a solids content of at least about 7 g/L. The MD-3 column is eluted with first and second MD-3 elution volumes that are made up of aqueous solvent. Either or both MD-3 elution volumes can be made up of gradient volumes that include different aqueous solvents or the composition of the aqueous solvent of either or both elution volumes may be varied linearly, exponentially, logarithymically, hyperbolically, or stepwise during elution of the respective elution volume. Effluents corresponding to the respective elution volumes or gradient volumes can be collected in toto or fractionate collected. Sixth products are obtained by removing aqueous solvent from the effluent or gradient fractions of a second MD-3 process, however collected.
An effluent or gradient fraction of a second MD-3 process is a starting material for making a sixth product that is at least about 85% by weight, 3,4xe2x80x2,5-trihydroxy-trans-stilbene in which the effluent or gradient fraction is concentrated to a concentrated composition and twice contacting this concentrated composition with separate extraction volumes of a volatile polar organic solvent (e.g., ethyl acetate), combining the extraction volumes, and removing the volatile polar organic solvent to obtain the 85% product. An alternative process for making the 85% product is provided in which an elution volume or gradient fraction from a second MD-3 process is evaporated to dryness, the residue so formed dissolved in water at a temperature greater than 0xc2x0 C. to form a solution, the solution cooled to less than about 0xc2x0 C. to form crystals, and separating the crystals from supernatant to obtain the 85% sixth product.
The present invention also provides a third MD-3 process for making a composition containing a seventh product that includes at least 50% by weight of 3,4xe2x80x2,5-trihydroxy-cis-stilbene. Starting material for a third MD-3 process is a fractionate collected gradient fraction of a second MD-2 process. The fractionate collected gradient fraction of a second MD-2 process is concentrated to a solids content of at least about 7 g/L to form a loading concentrate that is eluted through an MD-3 column. The third MD-3 process further includes the steps of eluting the MD-3 column with first, second, and third elution volumes that are made up of aqueous solvent. Each of the elution volumes can be made up of two or more gradient volumes in which the aqueous solvent has the same or a different composition. The elution volumes or gradient volumes result in MD-3 effluents of a third MD-3 process. The elution volumes or gradient volumes are collected to toto or fractionate collected. The effluents of the third MD-3 elution volume of a third MD-3 process, or gradient fractions thereof, are compositions containing the seventh product of the present invention. The seventh products of the present invention are obtained by removing aqueous solvent from the effluent resulting from the third MD-3 elution volume of a third MD-3 process, or gradient fractions thereof.
A process for converting a mono-xcex2-D-glycosylated trihydroxystilbene to the corresponding aglycone is also provided. The process includes the steps of providing a solution or suspension of a glycosolated trihydroxystilbene, contacting the solution or suspension with HCl at a total concentration between 0.01 and 0.02 g/ml, and refluxing the acidified solution or suspension for about 10 to about 200 minutes. The corresponding aglycone is isolated from the reaction mixture by techniques as are known in the art. The converting process can be carried out under a blanket of inert gas, for example, nitrogen.
Definitions
Alcohol. As used herein, the term alcohol refers to a lower aliphatic alcohol, in particular one selected from the group consisting of methanol, ethanol, the isomeric propanols, the isomeric butanols, the isomeric pentanols, and the isomeric hexanols.
Aqueous Solvent. As used herein, the term aqueous solvent refers to water or a polar organic solvent that is miscible with water in all proportions from 1:99 to 99:1. Examples of polar organic solvents that are, or can be used, as components of an aqueous solvent, as that term is herein used, includes but is not limited to methanol, ethanol, isopropanol, n-propanol, acetone, and acetonitrile. Other suitable polar organic solvents are known to the skilled artesian.
Column Volume. As used herein, column volume refers to the volume of the space defined by the inner surface of the chromatography column or chamber that surrounds the RPLC stationary phase. Column volume is abbreviated herein as CV.
Composition. As used herein, the term composition is a slurry, suspension, dispersion, or especially a solution of a material, normally solid at room temperature, in an aqueous solvent. Examples of compositions include but are not limited to loading elutions, loading concentrates, and effluents or gradient fractions from any reverse phase liquid chromatography process of the present invention.
Fractionate Collecting. When used in connection with an effluent or a gradient effluent, or a gradient fraction, the term fractionate collecting denotes that the effluent or gradient effluent or gradient fraction or gradient subfraction is segregated into at least two portions or aliquots.
MD-1 Column. An MD-1 column is a reverse phase liquid chromatography column of any size in which the stationary phase is a crosslinked copolymer of a vinyl aromatic compound, for example styrene, cross linked with a polyvinyl aromatic compound, for example divinylbenzene, wherein the stationary phase has a mean surface area of at least about a 400 m2/g, preferably 800 m2/g, and a porosity of at least about 0.55 ml/ml, preferably at least 0.58 ml/ml. The mean diameter of the particles comprising the stationary phase is between about 490xcexc and 700xcexc. The dipole moment of the crosslinked polymer comprising the stationary phase is less than about 0.5. Water is used for conditioning an MD-1 column.
MD-2 Column. An MD-2 column is a reverse phase liquid chromatography column of any size in which the stationary phase is a polyamide resin. As used herein, polyamide resin is a polymer of a lactam or a copolymer of a diamine and a dicarboxylic acid (or of the salt formed between the diacid and the diamine). Examples of polyamide resins include poly(caprolactam) and poly(hexamethylene adipamide). An alcohol water mixture comprising 10 vol-% methanol is used to condition an MD-2 column.
MD-3 Column. An MD-3 column is a reverse phase liquid chromatography column of any size in which the stationary phase is silica gel based reverse phase particles having C8 to C18, alkane moieties or cyano moieties bonded to its surface. A suitable material is WP Octadecyl reverse phase media available from J. T. Baker, Phillipsburg, N.J. (Cat. #7248-2). An MD-3 column is conditioned with a mixture of methanol and water comprising about 20 vol-% methanol.
Percent Solid. As used herein, the quantity percent solids refers to the weight of a nominally solid composition comprising an aqueous solvent that remains after the aqueous solvent is removed. Unless otherwise indicated, the quantity percent solid is expressed as the ratio of the weight of the composition remaining after removal of aqueous solvent divided by the weight of the composition before removal of the aqueous solvent, multiplied by 100. A nominally solid composition is a composition that does not flow under its own weight at room temperature.
Pharmaceutically acceptable processing excipients. Pharmaceutically acceptable processing excipients are pharmaceutically acceptable organic or inorganic carrier substances that do not react or otherwise interfere with biologically active components of pharmaceuticals or neutriceuticals and which assist in processing the biologically active components, or products containing them, into a form convenient for administering the biologically active components to an animal, including a human. Many such pharmaceutically acceptable processing excipients are known in the art. Among these, tricalcium phosphate and maltodextrin are particularly preferred.
SDA. As used herein SDA refers to specially denatured alcohol. See U.S. Pharmacopoeia.
Solids Component. The portion of a slurry, suspension, dispersion or solution is an aqueous solvent that remains after the aqueous solvent is removed. Synonymous with solids portion.
Solids Content. As used herein, the term solids content quantifies the portion of a solution, slurry, suspension, or dispersion in an aqueous solvent that remains when the aqueous solvent is removed and is expressed in units of grams of solid remaining per liter of solution or slurry and is abbreviated g/L.
Stilbene Fraction. Stilbene fraction refers collectively to the constituents or components of a material especially a solids component, that consists essentially of 1,2-diphenylethenes and substituted 1,2-diphenylethenes, where either or both of the phenyl rings can bear one or more substituents.
Volume Percent. As used herein the term volume percent, abbreviated vol-%/, is used to describe the composition of an aqueous solvent. The vol-% of a component represents the ratio of the volume of the component added to a composition to the total of the volumes of all components added to the composition times 100. The volume percent of an aqueous solvent can be easily calculated at the time it is formulated or it can be determined later using standard techniques, for example, GL chromatography using suitable reference mixtures.
In one embodiment, the present invention provides a first product that comprises at least about 60%, preferably at least about 65%, by weight solids which solids comprise at least about 10%, preferably at least about 12%, by weight of a stilbene fraction. According to the present invention, the first product can be obtained by providing a solid plant material, preferably V. vinifera, more preferably P. cuspidatum, which plant material has been cut or ground to pieces having an average volume from about 0.001 mm3 to about 15 mm3, and contacting the plant material with an aqueous solvent, preferably an alcohol-water mixture comprising about 75 volume percent (vol-%) SDA. The contacting may be by any suitable means as are known in the art; for example, percolation, vat extraction, counter current extraction, and the like. The first product can then be obtained by removing aqueous solvent or a component thereof from the resulting composition. In this or any embodiment, aqueous solvent can be removed by any of the means as are known in the art such as evaporation, distillation, and lyophilization, to mention a few.
The first product can be used to prepare a second product that can be directly administered to an animal, including a human, and which second product has a stilbene fraction amounting to at least about 8% by weight and at least one pharmaceutically acceptable processing excipient.
According to one embodiment of the present invention, the second product is made by slurrying the first product in water and homogenizing the slurry with one or more pharmaceutically acceptable processing excipients. A Silverson Model 14 RT-A homogenizer, Silverson Corporation, East Longmeadow, Mass. is suitable for this purpose. The homogenized mixture is then dried by spray drying or vacuum drying.
According to another embodiment of the invention, the first product is also useful as a starting material for preparation of products having a stilbene fraction of at least about 20% by weight or for the preparation of trihydroxystilbenes and glycosylated derivatives thereof by employing reverse phase liquid chromatography processes.
In reverse phase liquid chromatography (RPLC) as practiced in embodiments of the present invention, the column packing (stationary phase, or adsorbent) is non-polar, typically having a dipole moment of 3 or less. Silica gel that has been treated to provide it with a bonded surface layer that is paraffinic in nature is an example of a stationary phase for reverse phase chromatography. Silica gels having permanently bonded C8 to C18 alkyl groups are commercially available as a stationary phase. Reverse phase liquid chromatography columns are eluted with eluents of decreasing polarity which causes the more polar compounds loaded on a column to elute first.
Reverse phase liquid chromatography stationary phases of organic material are also known. Polymers of vinyl aromatic compounds, for example styrene, that are heavily crosslinked with polyvinylic aromatic hydrocarbons, for example divinylbenzene, can be used as stationary phases for reverse phase liquid chromatography. These organic polymeric stationary phases are made by processes that yield small, extremely rigid, macroreticular particles. Highly crosslinked acrylic polymers are also useful as stationary phases for reverse phase liquid chromatography. Suitable stationary organic phases are commercially available. For example, styrenic and acrylic stationary phases are available from the Rohm and Haas Company, Philadelphia, Pa., under the trade name Amberlite(copyright). Styreneic stationary phases are also available under the trade name Amberchrom(copyright) from Tossohass, Montgomeryville, Pa. Polyamide resins (e.g. nylons), polyester resins, and phenolic resins are also useful stationary phases for the reverse phase chromatography processes of the present invention.
Many polar organic solvents are suitable eluents for reverse phase liquid chromatography. Lower alcohols, such as methanol, ethanol, and propanol, as well as nitrites such as acetonitrile, are used as organic eluents. Lower aliphatic ketones such as acetone, methyl ethyl ketone, and diethyl ketone, as well as cyclic ethers such as tetrahydrofuran, can also be used. Dimethyl formamide, dimethyl sulfoxide, and alkyl esters of acetic acid such as ethyl acetate can also be used. Mixtures of such solvents in various proportions can be used when it is desired to elute or wash the column with solvents of varying polarity, from high to low relative polarity. Applicants have found that mixtures of water and an alcohol, for example, methanol, ethanol, n-propanol, iso-propanol, n-butanol, and n-and sec-hexanol, are particularly useful as mobile phases or eluents for separating and purifying stilbene compounds, especially those obtained from plant material. The RPLC processes of the present invention are advantageously carried out using an eluent of variable composition, that is a so-called gradient eluent. The limits of concentration of gradient eluents are determined by the concentration of polar organic solvent necessary to elute products from the stationary phase and by the requirement that the polar organic solvent be miscible to form a single phase at the required concentration.
In certain embodiments of the present invention the initial alcohol concentration is 10 volume percent (10 vol-%) or less and is increased as separation and purification proceeds.
The reverse phase liquid chromatography systems used to practice the present invention may be either preparative or analytical. Preparative columns require larger loading capacity and are typically larger in size.
Flow rates of the eluent are adjusted according to the column dimensions, the degree of separation desired, the particle size of the stationary phase, and the back pressure in the column. The separation is typically carried out at 20xc2x0 C. to 30xc2x0 C. However, a temperature up to about 45xc2x0 C. can be used. The separation may be carried out at high pressure (500-200 psi) or moderate pressures (100-500 psi) or, preferably, at lower pressures (10-100 psi).
With regards to the dimensions of the reverse phase liquid chromatographic column, the loading of the column, the temperature, and flow rate, one skilled in the art will know to vary these parameters based primarily upon practical considerations known in the art.
The product to be chromatographically treated is generally provided as a solution or suspension in an aqueous solvent. Preferably, the aqueous solvent is a mixture of an alcohol and water having a volume percent alcohol between about 5 vol-% and about 20 vol-%, as determined by known methods, for example gas chromatography. The concentration of product in the solution or suspension to be chromatographically treated is also varied according to the particular embodiment, but is generally between about 0.1 and about 10 g/L. Preferably, the concentration of the product to be treated is such that column loading is between about 1 g/L and 12 g/L.
The reverse phase liquid chromatography column can be conditioned by eluting the column with a conditioning volume of a conditioning liquid, preferably an aqueous solvent. The conditioning volume is preferably between about 1 and about 10 column volumes.
The product to be treated is loaded onto the conditioned stationary phase of the reverse phase chromatography column by means of a solution, a slurry, or, a loading concentrate obtained by evaporating an aqueous solvent, preferably alcohol, from a composition containing the product. Loading of the column is accomplished by eluting the solution, slurry, or loading concentrate through the column. Preferably, elution of the solution, slurry, or loading concentrate is followed by elution with a washing elution volume comprising an aqueous solvent having the same composition as the aqueous solvent of the solution, slurry, or loading concentrate used to load the column stationary phase. The washing elution volume, when one is used, is preferably between about I and about 10 column volumes.
A further embodiment of the present invention provides a third product from an MD-1 reverse phase liquid chromatography process having at least about 20% by weight and more preferably at least about 24% by weight of a mixture of trihydroxystilbenes and mono-xcex2-D-glycosylated trihydroxystilbenes. In a preferred embodiment of the MD-I process, the first product of the present invention is slurried in an aqueous solvent, preferably a mixture comprising between about 3 vol-% and about 7 vol-% alcohol, preferably methanol. The first product is loaded onto an MD-1 column support by eluting the slurry through the MD-1 column and can be followed by a washing elution volume including an aqueous solvent that is preferably a mixture of alcohol and water having between about 5 vol-% and about 20 vol-% alcohol, preferably methanol. A composition including the third product can be eluted from the loaded MD-1 column stationary phase with a first MD-1 elution volume to produce a first MD-1 effluent. The first MD-1 elution volume includes an aqueous solvent, preferably a mixture of alcohol and water having between about 70 vol-% and about 80 vol-%, preferably about 75 vol-%, of an alcohol, preferably a methanol. Aqueous solvent can be removed from the first MD-1 effluent composition by any suitable means as discussed above, to obtain the third product.
Yet another embodiment of the present invention provides a fourth product having at least about 30% by weight of a stilbene fraction having a mixture of trihydroxystilbenes and mono-xcex2-D glycosylated trihydroxystilbenes. The fourth product can be obtained by a first MD-2 reverse phase liquid chromatography process.
Starting material for a first MD-2 process is a first MD-2 loading concentrate having a third product of the present invention in an aqueous solvent, preferably a mixture of alcohol and water comprising not more than about 20 vol-% alcohol, preferably methanol. The MD-2 loading concentrate can be made by removing sufficient aqueous solvent from the first MD-1 effluent resulting from the first MD-1 elution volume so that the solids content of the first MD-2 loading concentrate is at least about 10 g/L, preferably at least about 13 g/L. The third product is loaded onto an MD-2 column stationary phase by eluting the first MD-2 loading concentrate through the MD-2 column which can be followed by a washing elution volume. The MD-2 column is then eluted with a first MD-2 elution volume to make a first MD-2 effluent. In one embodiment, the first MD-2 elution volume is a mixture of an alcohol, preferably methanol, and water having at least about 60 vol-% and preferably at least about 70 vol-% alcohol, more preferably at least about 75 vol-% alcohol.
In another embodiment, a second MD-2 process, which includes the steps of the first MD-2 process, the MD-2 column is eluted with a first MD-2 elution volume of a second MD-2 process that includes at least a first gradient volume and a second gradient volume, both of which are mixtures of an alcohol, preferably methanol and water, and both of, which can be divided into any number of subgradient volumes. In a preferred embodiment, the first gradient volume includes between about 20 vol-% and about 40 vol-%, preferably at least about 30 vol-%, of an alcohol, preferably methanol, and the second gradient volume includes between about 70 vol-% to about 80 vol-%, preferably at least about 75 vol-%, of an alcohol, preferably methanol.
In those embodiments of the second MD-2 process in which the first MD-2 elution volume of a second MD-2 process has first and second gradient volumes, the effluent that results from elution of the first and second gradient volumes can be fractionate collected and segregated into first and second gradient fractions, respectively, of the second MD-2 process that are compositions containing specific embodiments of the fourth product of the present invention. Furthermore, either gradient fraction can itself be fractionate collected to obtain gradient subfractions.
In preferred embodiments of the second MD-2 process in which the first gradient volume is a mixture of alcohol and water having about 30 vol-% methanol and the second gradient volume is a mixture of alcohol and water having about 70 vol-% methanol, the first gradient fraction is a composition including a fourth product of the present invention that includes at least about 40% by weight of a stilbene fraction that includes at least about 90% mono-xcex2-D-glycosylated trihydroxystilbenes and the second gradient fraction is a composition also including a fourth product of the present invention including at least about 30% by weight of a stilbene fraction that has at least about 80% trihydroxystilbenes, preferably 3,4xe2x80x2,5-trihydroxystilbenes.
The respective fourth products can be obtained by removing alcohol-water mixture from the respective gradient fractions.
Another embodiment of the present invention provides a third MD-2 process for making a composition that includes a stilbene fraction that has at least about 80% and preferably at least about 90% by weight mono-xcex2-D-glycosylated-3,4xe2x80x2,5-trihydroxystilbene. In a third MD-2 process, a third product is loaded onto an MD-2 column stationary phase by means of an MD-2 loading concentrate. The MD-2 column is eluted with a first MD-2 elution volume of a third MD-2 process. In a preferred embodiment, the first MD-2 elution volume of a third MD-2 process is an MD-2 gradient elution volume including a mixture of alcohol and water the composition of which can be varied linearly, exponentially, logarithmically, parabolically, step-wise, or according to any combination of the foregoing. The MD-2 effluent is fractionate collected to obtain one or more compositions, each of which contains a fourth product.
Another embodiment of the present invention provides a fifth product that has at least about 60%, preferably at least about 65%, of a stilbene fraction containing at least about 90% by weight of mono-xcex2-D-glycosylated-3,4xe2x80x2,5-trihydroxy-trans-stilbene. This embodiment of the fifth product of the present invention can be made in a first MD-3 reverse phase chromatography process. Starting material for this first MD-3 reverse phase chromatography process is a loading concentrate made by removing sufficient aqueous solvent from the segregated first gradient fraction of the second MD-2 process or a segregated fraction of the third MD-2 process that includes a stilbene fraction that has at least about 50% of mono-xcex2-D-glycosylated-3,4xe2x80x25-trihydroxystilbenes, so that the loading concentrate has a solids content of at least about 3 g/L. In preferred embodiments in which the first gradient volume of the second MD-2 elution volume is a mixture of alcohol and water, the loading concentrate preferably has not more than about 5% alcohol. The loading concentrate is eluted through an MD-3 column to load the column stationary phase and, in preferred embodiments, is followed by a washing elution that is an aqueous solvent, preferably a mixture of alcohol and water having about 5 vol-% alcohol, preferably methanol, and the volume of the loading elution corresponds to about 0.5 to about 10 column volumes. The MD-3 column is then eluted with a first MD-3 elution volume of the first MD-3 process to obtain a first MD-3 effluent of a first MD-3 process that is fractionate collected to obtain a first fraction of a first MD-3 effluent of a first MD-3 process and a second fraction of a first MD-3 effluent of a first MD-3 process. In preferred embodiments the first fraction of the first MD-3 effluent of a first MD-3 process amounts to about 0.5 to about 3, preferably about 1.5, column volumes and the second fraction of the first MD-3 effluent of a first MD-3 process amounts to between about 0.5 and about 3 column volumes, preferably 1 column volume. The fifth product that has at least about 60% of a stilbene fraction comprising at least about 90% mono-xcex2-D-glycosylated-3,4xe2x80x2,5-trihydroxy-trans-stilbene can be obtained by removing the aqueous solvent from the fractionate collected first MD-3 effluent of a first MD-3 process.
In another embodiment, the present invention provides an evaporative crystallization process for making a fifth product containing at least about 85% and preferably at least about 90% by weight 3,4xe2x80x2,5-trihydroxy-trans-stilbene-3-xcex2-mono-D-glucoside. The starting point for the evaporative crystallization process is fractionate collected first MD-3 effluent of a first MD-3 process, preferably a second fraction of a first MD-3 effluent of a first MD-3 process that is fractionate collected after a first fraction of a first MD-3 effluent of a first MD-3 process amounting to 0.5 to about 3 column volumes is collected. The second fraction of a first MD-3 effluent of a first MD-3 process is evaporated to between about 0.1 and about 0.2 times its original volume and cooled, preferably to 4xc2x0 C. or below, to form crystals that are a fifth product containing at least about 85% 3,4xe2x80x2,5-trihydroxy-trans-stilbene-3-xcex2-mono-D-glucoside.
In another embodiment, the present invention provides a sixth product having at least about 70% and preferably at least about 75% of a stilbene fraction including at least about 70% by weight of 3,4xe2x80x2,5-trihydroxy-trans-stilbene. The sixth product can be prepared by a second MD-3 process. The starting material for the second MD-3 process is the second gradient fraction of the second MD-2 process. Aqueous solvent is removed from the second MD-2 gradient fraction of the second MD-2 process to form a loading concentrate having a solids content of at least about 7 g/L. The loading concentrate is eluted through an MD-3 column and, in preferred embodiments, can be followed by a washing elution volume including an aqueous solvent, preferably a mixture of alcohol and water including between about 10 vol-% and about 20 vol-% alcohol, preferably methanol. The MD-3 column is then eluted with a first MD-3 elution volume of a second MD-3 process having first and second gradient volumes. The first gradient volume of the first MD-3 elution volume of the second MD-3 process is preferably an aqueous solvent that is preferably a mixture of alcohol and water having between about 35 vol-% and about 45 vol-%, preferably 40 vol-%, of an alcohol, preferably methanol, and elutes a first MD-3 gradient fraction of a second MD-3 process and is followed by elution with the second MD-3 gradient volume of a first MD-3 elution volume of the second MD-3 process that includes an aqueous solvent, preferably an alcohol-water mixture having between about 50 vol-% and about 60 vol-%, preferably about 65 vol-%, of an alcohol, preferably methanol, to elute a second MD-3 gradient fraction of the second MD-3 process. The sixth product of the present invention can be obtained by removing the aqueous solvent from the second MD-3 gradient fraction of the second MD-3 process.
In other embodiments, the present invention provides a sixth product that includes at least about 85% and preferably at least about 90% trans-resveratrol (3,4xe2x80x2,5-trihydroxy-trans-stilbene) which can be obtained by an extraction process. In one embodiment, the extraction process includes removing aqueous solvent from the second MD-3 gradient fraction of a second MD-3 process to attain a solids content of at least about 1.5 g/L and twice contacting the so concentrated second MD-3 gradient volume with one or more extraction volumes, each preferably 0.5 to 2 times the volume of the so concentrated second MD-3 gradient volume, of a polar organic solvent, preferably ethyl acetate. The extraction volumes are combined and the polar organic solvent is removed to obtain the sixth product having at least about 85% by weight trans-resveratrol.
Yet another embodiment of the present invention is a crystallization process for making the substantially colorless product which comprises removing the aqueous solvent from the second MD-3 gradient volume of a first MD-3 elution volume of a second MD-3 process, dissolving the resulting solid at T greater than 10xc2x0 C. in methanol, cooling to T less than 0xc2x0 C. form crystals of the substantially colorless product and recovering the crystals of substantially colorless product by conventional means.
In another embodiment, the invention provides a partition crystallization process for making a sixth product that contains at least about 80% and preferably at least about 85% 3,4xe2x80x2,5-trihydroxy-trans-stilbene. Starting point for the partition crystallization process is a second MD-3 gradient fraction of a second MD-3 process. The second MD-3 gradient fraction is concentrated under vacuum to 0.35 to 0.40 times its original volume and a solid concentration of at least about 1,5 g/L. The concentrated gradient fraction is contacted with a polar organic solvent, preferably ethyl acetate. Preferably, the volume of the polar organic solvent used is between bout 0.75 and about 0.85 time the volume of the concentrated second gradient fraction. In preferred embodiments, the gradient fraction is contacted serially with two separate volumes of polar organic solvent and the volumes are combined. The polar organic solvent, from single or multiple contactings, are evaporated to dryness to yield a sixth product having at least 80% 3,4xe2x80x2,5-trihydroxy-trans-stilbene.
Yet another embodiment of the present invention provides a seventh product that has at least about 50% and preferably at least about 55% more preferably at least about 60%, by weight of a stilbene fraction that includes at least about 50% by weight of 3,4xe2x80x2,5-trihydroxy-cis-stilbene.
The seventh product of the present invention can be prepared by a third MD-3 reverse phase liquid chromatography process. Starting material for the third MD-3 process is a second MD-2 gradient fraction of a second MD-2 process from which aqueous solvent is removed to form a loading concentrate having a solid content of at least 7 g/L. The loading concentrate thus formed is eluted through a conditioned MD-3 column. In preferred embodiments, elution of the loading concentrate can be followed by elution of a washing elution volume including an aqueous solvent, preferably a mixture of alcohol and water having between about 5 vol-% and about 20 vol-% alcohol, preferably methanol. The washing elution volume, when used, is followed by first and second MD-3 elution volumes of the third MD-3 process to produce, respectively, first and second effluents of the third MD-3 process. The first MD-3 elution volume of the third MD-3 process can include an aqueous solvent of a particular composition and can have two or more gradient volumes that when fractionate collected, result in two or more gradient fractions of a first effluent of the third MD-3 process. The first and second MD-3 elution volumes of the third MD-3 process include aqueous solvents which, in preferred embodiments, are mixtures of alcohol and water.
The first MD-3 elution volume of the third MD-3 process preferably includes a mixture of alcohol and water comprising up to about 70% alcohol, preferably methanol. In one embodiment, the first MD-3 elution volume of the third MD-3 process comprises first and second gradient volumes of a first MD-3 elution volume of a third MD-3 process that are mixtures of alcohol, preferably methanol, and water wherein the first MD-3 gradient volume of a first MD-3 elution volume of a third MD-3 process has between about 35 vol-% and about 45 vol-%, preferably about 40 vol-%, alcohol and the second gradient volume of a first MD-3 elution volume of a third MD-3 process comprises between about 50 vol-% and about 60 vol-%, preferably about 55 vol-%, alcohol. In other embodiments, the first elution volume of the third MD-3 process is a gradient elution volume and includes an aqueous solvent the composition of which is varied over the course elution of the first elution volume of a third MD-3 process according to a predetermined program. The program may be linear, exponential, logarithmic, hyperbolic, step-wise, or a combination of the foregoing. For example, if the aqueous solvent is a mixture of alcohol and water, the volume percent alcohol can be varied from about 20 vol-% to about 60 vol-% during elution of the first MD-3 elution volume of the third MD-3 process.
The volume of this first MD-3 elution volume of the third MD-3 process is from about 1 to about 12 column volumes, preferably less than about 8 column volumes.
The second elution volume of the third MD-3 process is preferably a mixture of an alcohol, preferably methanol, and water including between about 80 vol-% and about 90 vol-%, preferably about 75 vol-% of alcohol. The seventh product can be obtained by collecting a second MD-3 effluent of a third MD-3 process eluted by the second MD-3 elution volume of the third MD-3 process and removing the aqueous solvent therefrom.
The second MD-3 effluent of a third MD-3 process eluted by the second MD-3 elution volume of a third MD-3 process can be fractionate collected. When the second MD-3 effluent of a third MD-3 process is fractionate collected, it may be collected in any number of fractions. In a preferred embodiment, the second effluent of the third MD-3 process is fractionate collected in two fractions. The first fraction of the second effluent of the third MD-3 process preferably amounts to between about 0.5 and about 1 column volume. The second fraction of the second effluent of the third MD-3 process preferably amounts to between about 0.5 and 2.0 column volumes and is a composition including the seventh product of the present invention.